This invention relates generally to organic chemistry, biochemistry, pharmacology and medicine. More particularly, it relates to geometrically restricted 2-indolinone derivatives and their physiologically acceptable salts and prodrugs which modulate the activity of protein kinases (xe2x80x9cPKsxe2x80x9d) and, therefore, are expected to exhibit a salutary effect against disorders related to abnormal PK activity.
The following is offered as background information only and is not admitted to be prior art to the present invention.
PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation, i.e., virtually all aspects of cell life in one way or another depend on PK activity. Furthermore, abnormal PK activity has been related to a host of disorders, ranging from relatively non-life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer).
The PKs can be conveniently broken down into two classes, the protein tyrosine kinases (PTKS) and the serine-threonine kinases (STKs).
One of the prime aspects of PTK activity is their involvement with growth factor receptors. Growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, growth factor receptors are converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor and other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules that, in turn, effect numerous cellular responses such as cell division (proliferation), cell differentiation, cell growth, expression of metabolic effects to the extracellular microenvironment, etc. For a more complete discussion, see Schlessinger and Ullrich, Neuron, 9:303-391 (1992) which is incorporated by reference, including any drawings, as if fully set forth herein.
Growth factor receptors with PTK activity are known as receptor tyrosine kinases (xe2x80x9cRTKsxe2x80x9d). They comprise a large family of transmembrane receptors with diverse biological activity. At present, at least nineteen (19) distinct subfamilies of RTKs have been identified. An example of these is the subfamily designated the xe2x80x9cHERxe2x80x9d RTKs, which include EGFR (epithelial growth factor receptor), HER2, HER3 and HER4. These RTKs consist of an extracellular glycosylated ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins.
Another RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR). IR and IGF-LR interact with insulin, IGF-I and IGF-II to form a heterotetramer of two entirely extracellular glycosylated a subunits and two xcex2 subunits which cross the cell membrane and which contain the tyrosine kinase domain.
A third RTK subfamily is referred to as the platelet derived growth factor receptor (xe2x80x9cPDGFRxe2x80x9d) group, which includes PDGFRxcex1, PDGFRxcex2, CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences.
Another group which, because of its similarity to the PDGFR subfamily, is sometimes subsumed into the later group is the fetus liver kinase (xe2x80x9cflkxe2x80x9d) receptor subfamily. This group is believed to be made up of kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1, VEGF-R2), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).
A further member of the tyrosine kinase growth factor receptor family is the fibroblast growth factor (xe2x80x9cFGFxe2x80x9d) receptor subgroup. This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well defined, it appears that the receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the tyrosine kinase sequence is interrupted by regions of unrelated amino acid sequences.
Still another member of the tyrosine kinase growth factor receptor family is the vascular endothelial growth factor (xe2x80x9cVEGFxe2x80x9d) receptor subgroup. VEGF is a dimeric glycoprotein similar to PDGF but has different biological functions and target cell specificity in vivo. In particular, VEGF is presently thought to play an essential role is vasculogenesis and angiogenesis.
A more complete listing of the known RTK subfamilies is described in Plowman et al., DNandP, 7(6):334-339 (1994) which is incorporated by reference, including any drawings, as if fully set forth herein.
In addition to the RTKS, there also exists a family of entirely intracellular PTKs called xe2x80x9cnon-receptor tyrosine kinasesxe2x80x9d or xe2x80x9ccellular tyrosine kinases.xe2x80x9d This latter designation, abbreviated xe2x80x9cCTK,xe2x80x9d will be used herein. CTKs do not contain extracellular and transmembrane domains. At present, over 24 CTKS in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detailed discussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which is incorporated by reference, including any drawings, as if fully set forth herein.
The serine/threonine kinases, STKs, like the CTKs, are predominantly intracellular although there are a few receptor kinases of the STK type. STKs are the most common of the cytosolic kinases; i.e., kinases that perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton. The cytosol is the region within the cell where much of the cell""s intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes.
RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer. Other pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, immunological disorders such as autoimmune disease, cardiovascular disease such as atherosclerosis and a variety of renal disorders.
With regard to cancer, two of the major hypotheses advanced to explain the excessive cellular proliferation that drives tumor development relate to functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto-oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
In view of the apparent link between PK-related cellular activities and wide variety of human disorders, it is no surprise that a great deal of effort is being expended in an attempt to identify ways to modulate PK activity. Some of this effort has involved biomimetic approaches using large molecules patterned on those involved in the actual cellular processes (e.g., mutant ligands (U.S. Pat. No. 4,966,849); soluble receptors and antibodies (App. No. WO 94/10202, Kendall and Thomas, Proc. Nat""l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature, 362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys., 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).
In addition to the above, attempts have been made to identify small molecules which act as PK inhibitors. For example, bis-monocylic, bicyclic and heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have been described as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), quinazoline derivatives (EP App. No. 0 566 266 A1), selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have all been described as PTK inhibitors useful in the treatment of cancer.
Our own efforts to identify small organic molecules which modulate PK activity and which, therefore, would be expected to be useful in the treatment and prevention of disorders driven by abnormal PK activity, has led us to the discovery of a family of novel geometrically restricted 2-indolinone derivatives which exhibit PK modulating ability and which are the subject of this invention.
Thus, the present invention relates generally to novel geometrically restricted 2-indolinone derivatives and their prodrugs and physiologically acceptable salts which modulate the activity of receptor tyrosine kinases (RTKs), non-receptor protein tyrosine kinases (CTKs) and serine/threonine protein kinases (STKs). In addition, the present invention relates to -the preparation and use of pharmaceutical compositions of the disclosed compounds and their physiologically acceptable salts and prodrugs in the treatment or prevention of PK driven disorders such as, by way of example and not limitation, cancer, diabetes, hepatic cirrhosis, cardiovasacular disease such as atherosclerosis, angiogenesis, immunological disorders such as autoimmune disease and renal disease.
The terms xe2x80x9c2-indolinone,xe2x80x9d xe2x80x9cindolin-2-one,xe2x80x9d xe2x80x9c2-oxindolexe2x80x9d and xe2x80x9coxindolexe2x80x9d are used interchangably herein; all refer to a chemical compound having the general structure: 
As used herein, the above terms are deemed to include sulfur derivative; i.e., when Z=sulfur.
xe2x80x9cGeometrically restrictedxe2x80x9d refers to the chemical structure about a double bond wherein groups attached to the double bond are set in their spatial relationship to one another by the very nature of the double bond. That is, atoms attached to a double bond must be coplanar; i.e., in the same plane as the atoms of the double bond itself. This is best demonstrated insofar as the compounds of this invention are concerned by looking at the generic structures shown in Formulas 1 and 2: 
Formula 1 represents a backbone structure of a compound of this invention. It is understood that 1 is presented by way of example only and not limitation; backbone structures other than that shown in 1 are within the scope and spirit of this invention. The point to be gleaned from 1 is the relationship of the atoms attached to the double bond at the 3-position of the indolinone. In 1, ring system a and ring system b are linked to the double bond through a ring carbon. Since the atoms to either side of the linking carbon atom must be coplanar due to the double bond, and since the rings themselves are internally co-planar, the entire molecule, ring systems a and b and the double bond, are co-planar. This is in contrast to Formula 2, wherein a single bond connects ring system axe2x80x2 to the double bond. Ring system axe2x80x2 is therefore free to rotate about the single bond permitting the ring systems axe2x80x2 and bxe2x80x2 to be non-co-planar, potentially even being perpendicular to one another.
A xe2x80x9cpharmacological compositionxe2x80x9d refers to a mixture of one or more of the compounds described herein, or physiologically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and/or excipients. The purpose of a pharmacological composition is to facilitate administration of a compound to an organism.
As used herein, a xe2x80x9cphysiologically acceptable carrierxe2x80x9d refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
An xe2x80x9cexcipientxe2x80x9d refers to an inert substance added to a pharmacological composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
A xe2x80x9cprodrugxe2x80x9d refers to an agent which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. An example, without limitation, of a prodrug would be a compound of the present invention which is administered as an ester (the xe2x80x9cprodrugxe2x80x9d) to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial.
A further example of a prodrug might be a short polypeptide, for example, without limitation, a 2-10 amino acid polypeptide, bonded through a terminal amino group to a carboxy group of a compound of this invention wherein the polypeptide is hydrolyzed or metabolized in vivo to release the active molecule.
General Structural Features.
In one aspect, the present invention relates to a geometrically restricted 2-indolinone compound having chemical structure I, II or III: 
The scope of this invention includes physiologically acceptable salts and prodrugs of the compound claimed herein.
R1 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, hydroxy, alkoxy, xe2x80x94C(xe2x95x90O)ORxe2x80x3, Rxe2x80x3C(xe2x95x90O)Oxe2x80x94, C-amido, C-thioamido, acetyl, xe2x80x94S(xe2x95x90O)2Rxe2x80x3, and trihalomethylsulfonyl, wherein
Rxe2x80x3 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon);
A, B, D and E are independently selected from the group consisting of carbon and nitrogen, wherein
when A, B, D or E is nitrogen, R2, R3, R4 or R5, respectively, does not exist;
F, G, J and K are independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur, wherein
when n is 1 and F, G, J or K is an atom other than carbon, R6, R7, R8 or R9, respectively, does not exist;
when n is 0 and F, G or K is oxygen or sulfur, R6, R8 or R9, respectively, does not exist.
R2, R3, R4, R5, R6, R7, R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, xe2x80x94S(xe2x95x90O)Rxe2x80x3, xe2x80x94S(xe2x95x90O)2Rxe2x80x3, S-sulfonamido, N-sulfonamido, N-trihalo-methanesulfonamido, xe2x80x94C(xe2x95x90O)Rxe2x80x3, xe2x80x94C(xe2x95x90O)ORxe2x80x3, Rxe2x80x3C(xe2x95x90O)Oxe2x80x94, cyano, nitro, halo, cyanato, isocyanato, thiocyanato, isothiocyanato, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and xe2x80x94NR13R14;
R2 and R3 or R3 and R4 or R4 and R5 or R6 and R7 or R7 and R8 or R8 and R9 may combine to form a methylenedioxy or an ethylenedioxy group;
R13 and R14 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, xe2x80x94C(xe2x95x90O)Rxe2x80x3, acetyl, xe2x80x94S(O)2Rxe2x80x3, trihalomethane-sulfonyl and, combined, a five-member or a six-member heteroalicyclic ring;
R10, R11 and R12 are independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, halo, cyano, trihalomethyl, hydroxy, alkoxy, alkylthio, aryloxy, arylthio, Rxe2x80x3C(xe2x95x90O)Oxe2x80x94, xe2x80x94C(xe2x95x90O)ORxe2x80x3, xe2x80x94C(xe2x95x90O)Oxe2x88x92M+, xe2x80x94(CH2)C(xe2x95x90O)ORxe2x80x3, xe2x80x94(CH2)rC(xe2x95x90O)Oxe2x88x92M+, C-amido, N-amido, cyanato, isocyanato, thiocyanato, isothiocyanato, amino, xe2x80x94S(xe2x95x90O)Rxe2x80x3, xe2x80x94S(xe2x95x90O)2Rxe2x80x3, nitro and xe2x80x94NR13R14;
R10 and R11 or R11 and R12 may combine to form an endo double bond;
Z is selected from the group consisting of oxygen and sulfur; r is 1, 2, 3, 4, 5, or 6; and,
n is 0 or 1.
The term xe2x80x9cendoxe2x80x9d refers to a double bond contained within a ring structure; for example, the double bond in the following structure is an xe2x80x9cendoxe2x80x9d double bond: 
As used herein, the term xe2x80x9calkylxe2x80x9d refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as xe2x80x9c1 to 20xe2x80x9d refers to each integer in the given range; e.g., xe2x80x9c1 to 20 carbon atomsxe2x80x9d means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms). More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more individually selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, cyanato, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, amino and xe2x80x94NR13R14, R13 and R14 being as defined above.
A xe2x80x9ccycloalkylxe2x80x9d group refers to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and, cycloheptatriene. A cycloalkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more individually selected from alkyl, aryl, heteroaryl, heteroalycyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, carboxy, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro, amino and xe2x80x94NR13R14, with R13 and R14 being as defined above.
An xe2x80x9calkenylxe2x80x9d group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond.
An xe2x80x9calkynylxe2x80x9d group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond.
An xe2x80x9carylxe2x80x9d group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, trihalo-methanesulfonamido, amino and xe2x80x94NR13R 14, R13 and R14 being as defined above.
As used herein, a xe2x80x9cheteroarylxe2x80x9d group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole. The heteroaryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, carboxy, sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and xe2x80x94NR13R14, R13 and R14 being defined above.
A xe2x80x9cheteroalicyclicxe2x80x9d group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic ring may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from alkyl, cycloaklyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thio-carbamyl, sulfinyl, sulfonyl, C-amido, N-amido, amino and xe2x80x94NR13R14, with R13 and R14 being as defined above.
A xe2x80x9chydroxyxe2x80x9d group refers to an xe2x80x94OH group.
An xe2x80x9calkoxyxe2x80x9d group refers to both an xe2x80x94O-alkyl and an xe2x80x94O-cycloalkyl group, as defined herein.
An xe2x80x9caryloxyxe2x80x9d group refers to both an xe2x80x94O-aryl and an xe2x80x94O-heteroaryl group, as defined herein.
A xe2x80x9cmercaptoxe2x80x9d group refers to an xe2x80x94SH group.
A xe2x80x9calkylthioxe2x80x9d group refers to both an S-alkyl and an xe2x80x94S-cycloalkyl group, as defined herein.
A xe2x80x9carylthioxe2x80x9d group refers to both an xe2x80x94S-aryl and an xe2x80x94S-heteroaryl group, as defined herein.
A xe2x80x9ccarbonylxe2x80x9d group refers to a xe2x80x94C(xe2x95x90O)Rxe2x80x3 group, where Rxe2x80x3 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as defined herein.
An xe2x80x9caldehydexe2x80x9d group refers to a carbonyl group where Rxe2x80x3 is hydrogen.
A xe2x80x9ccycloketonexe2x80x9d refer to a cycloalkyl group in which one of the carbon atoms which form the ring has a xe2x80x9cxe2x95x90Oxe2x80x9d bonded to it; i.e. one of the ring carbon atoms is a xe2x80x94C(xe2x95x90O)-group.
A xe2x80x9cthiocarbonylxe2x80x9d group refers to a xe2x80x94C(xe2x95x90S)Rxe2x80x3 group, with Rxe2x80x3 as defined herein.
An xe2x80x9cO-carboxyxe2x80x9d group refers to a Rxe2x80x3C(xe2x95x90O)O-group, with Rxe2x80x3 as defined herein.
A xe2x80x9cC-carboxyxe2x80x9d group refers to a xe2x80x94C(xe2x95x90O)ORxe2x80x3 groups with Rxe2x80x3 as defined herein.
As used herein, an xe2x80x9cesterxe2x80x9d is a C-carboxy group, as defined herein, wherein Rxe2x80x3 is any of the listed groups other than hydrogen.
A xe2x80x9cC-carboxy saltxe2x80x9d refers to a xe2x80x94C(xe2x95x90O)Oxe2x88x92M+ group wherein M+ is selected from the group consisting of lithium, sodium, magnesium, calcium, potassium, barium, iron, zinc and quaternary ammonium.
An xe2x80x9cacetylxe2x80x9d group refers to a xe2x80x94C(xe2x95x90O)CH3 group.
A xe2x80x9ccarboxyalkylxe2x80x9d group refers to xe2x80x94(CH2)rC(xe2x95x90O)ORxe2x80x3 wherein r is 1-6 and Rxe2x80x3 is as defined above.
A xe2x80x9ccarboxyalkyl saltxe2x80x9d refers to a xe2x80x94(CH2)rC(xe2x95x90O)Oxe2x88x92M+ wherein M+ is selected from the group consisting of lithium, sodium, potassium, calcium, magnesium, barium, iron, zinc and quaternary ammonium.
A xe2x80x9ccarboxylic acidxe2x80x9d group refers to a C-carboxy group in which Rxe2x80x3 is hydrogen.
A xe2x80x9chaloxe2x80x9d group refers to fluorine, chlorine, bromine or iodine.
A xe2x80x9ctrihalomethylxe2x80x9d group refers to a xe2x80x94CX3 group wherein X is a halo group as defined herein.
A xe2x80x9ctrihalomethanesulfonylxe2x80x9d group refers to a X3CS(xe2x95x90O)2-group with X as defined above.
A xe2x80x9ccyanoxe2x80x9d group refers to a xe2x80x94Cxe2x95x90xe2x80x94N group.
A xe2x80x9ccyanatoxe2x80x9d group refers to a xe2x80x94CNO group.
An xe2x80x9cisocyanatoxe2x80x9d group refers to a xe2x80x94NCO group.
A xe2x80x9cthiocyanatoxe2x80x9d group refers to a xe2x80x94CNS group.
An xe2x80x9cisothiocyanatoxe2x80x9d group refers to a xe2x80x94NCS group.
A xe2x80x9csulfinylxe2x80x9d group refers to a xe2x80x94S(xe2x95x90O)Rxe2x80x3 group, with Rxe2x80x3 as defined herein.
A xe2x80x9csulfonylxe2x80x9d group refers to a xe2x80x94S(xe2x95x90O)2Rxe2x80x3 group, with Rxe2x80x3 as defined herein.
A xe2x80x9csulfonamidoxe2x80x9d group refers to a xe2x80x94S(xe2x95x90O)2NR13R14, with Rxe2x80x3 and R14 as defined herein.
A xe2x80x9ctrihalomethanesulfonamidoxe2x80x9d group refers to a X3CS(xe2x95x90O)2NR13-group with X and R13 as defined herein.
An xe2x80x9cO-carbamylxe2x80x9d group refers to a xe2x80x94OC(xe2x95x90O)NR13R14 group with R13 and R14 as defined herein.
An xe2x80x9cN-carbamylxe2x80x9d group refers to a R14OC(xe2x95x90O)NR13xe2x80x94 group, with R13 and R14 as defined herein.
An xe2x80x9cO-thiocarbamylxe2x80x9d group refers to a xe2x80x94OC(xe2x95x90S)NR13R14 group with R13 and R14 as defined herein.
An xe2x80x9cN-thiocarbamylxe2x80x9d group refers to a R14OC(xe2x95x90S)NR13xe2x80x94 group, with R13 and R14 as defined herein.
An xe2x80x9caminoxe2x80x9d group refers to an xe2x80x94NR13R14 group, with R13 and R14 both being hydrogen.
A xe2x80x9cC-amidoxe2x80x9d group refers to a xe2x80x94C(xe2x95x90O)NR13R14 group with R13 and R14 as defined herein. An xe2x80x9cN-amidoxe2x80x9d group refers to a R13C(xe2x95x90O)NR14xe2x80x94 group with R13 and R14 as defined herein.
A xe2x80x9cnitroxe2x80x9d group refers to a xe2x80x94NO2 group.
A xe2x80x9cquaternary ammoniumxe2x80x9d group refers to a xe2x80x94+NR13R14R15 group wherein R13R14 and R15 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl.
A xe2x80x9cmethylenedioxyxe2x80x9d group refers to a xe2x80x94OCH2Oxe2x80x94 group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.
An xe2x80x9cethylenedioxyxe2x80x9d group refers to a xe2x80x94OCH2CH2Oxe2x80x94 group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.
Another aspect of this invention is a combinatorial library of at least 10 compounds formed by reacting an oxindole having the general chemical structure: 
with a cycloketone having one of the general chemical structures: 
wherein A, B, D, E, F, G, J, K, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are as previously defined herein.
By xe2x80x9creactingxe2x80x9d is meant placing one of the above oxindoles and one of the above cycloketones in a chemical environment wherein they will interact with on another to form a covalent bond between them. In the present case, the covalent bond will ultimately be a double bond from the carbon atom at the 3-position of the oxindole to the keto carbon atom of the cycloketone.
A xe2x80x9ccombinatorial libraryxe2x80x9d refers to all the compounds formed by the reaction of each compound in one dimension of a multi-dimensional array with a compound in each of the other dimensions of the multi-dimensional array. As used herein, the multi-dimensional array is two-dimensional, one dimension being all the oxindoles of this invention, the other dimension being all the cycloketones of this invention. Each oxindole may be reacted with each of the cycloketones to form a 2-indolinone. All 2-indolinone compounds formed in this manner are within the scope of this invention. Also within the scope of this invention are smaller combinatorial libraries formed by the reaction of some of the oxindoles of this invention with all of the cycloketones of this invention or all of the oxindoles with some of the cycloketones or some of the oxindoles with some of the cycloketones.
It is another aspect of this invention that a combinatorial library may be used to screen compounds of this invention for a desired activity.
By a xe2x80x9cdesired activityxe2x80x9d is meant the ability to modulate the catalytic activity of a selected protein kinase.
By xe2x80x9cscreeningxe2x80x9d is meant to contact an entire combinatorial library of compounds or any portion thereof with one or more target protein kinases and then observe the effect of the compounds on the catalytic activity of the protein kinase.
Yet another aspect of this invention is a compound which modulates protein kinase activity, in particular RTK, CTK or STK kinase catalytic activity.
Preferred Structural Features.
A presently preferred embodiment of this invention is a compound in which:
n is 1;
A, B, D and E are carbon;
R1 is hydrogen; and,
Z is oxygen.
A further presently preferred embodiment of this invention is one in which:
n is 1;
A, B, D and E are carbon;
R1 is hydrogen;
Z is oxygen; and,
F, G, J and K are carbon.
Still another presently preferred embodiment of this invention is a compound in which:
n is 0;
A, B, D and E are carbon; and,
Z is oxygen.
A compound in which:
n is 0;
A, B, D and E are carbon;
Z is oxygen;
F is nitrogen;
R9 is hydrogen; and,
G and K are carbon
is yet another presently preferred embodiment of this invention.
A presently preferred embodiment of this invention would also be a compound in which:
n is 0;
A, B, D and E are carbon;
Z is oxygen;
K is nitrogen;
R6 is hydrogen; and,
F and G are carbon.
A further presently preferred embodiment of this invention is a compound in which one or two of F, G, J or K are independently nitrogen.
It is likewise a presently preferred embodiment of this invention that
n is 1;
A, B, D and E are carbon;
R1 is hydrogen;
Z is oxygen;
R13 is hydrogen; and,
R14 is unsubstituted lower alkyl.
It is still another presently preferred embodiment of this invention that:
R2, R3, R4, R5, R6, R7, R8 and R9 are independently selected from the group consisting of:
hydrogen;
unsubstituted lower alkyl;
lower alkyl substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3 and xe2x80x94NR13R14;
unsubstituted lower alkoxy;
lower alkoxy substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3, unsubstituted aryl or xe2x80x94NR13R14;
trihalomethyl;
unsubstituted alkenyl;
unsubstituted alkynyl;
unsubstituted aryl;
aryl substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl or lower alkyl substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3 and xe2x80x94NR13R14;
unsubstituted heteroalicyclic;
heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, xe2x80x94C(xe2x95x90O)H, xe2x80x94C(xe2x95x90O)xe2x80x94 (unsubstituted lower alkyl), hydroxy, unsubstituted alkoxy, alkoxy substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3 and xe2x80x94NR13R14;
unsubstituted aryloxy;
aryloxy substituted with a group independently selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, hydroxy and amino;
mercapto;
unsubstituted alkylthio;
unsubstituted arylthio;
arylthio substituted with one or more groups independently selected from the group consisting of halo, hydroxy and amino;
S-sulfonamido;
xe2x80x94C)xe2x95x90O)ORxe2x80x3;
Rxe2x80x3C(xe2x95x90O)Oxe2x80x94;
hydroxy;
cyano;
nitro;
halo;
C-amido;
N-amido;
amino; and,
xe2x80x94NR13R14.
A compound in which:
n is 1;
A, B, D and E are carbon;
R1 is hydrogen;
Z is oxygen;
R2, R3, R4, R5, R6, R7, R8 and R9 are independently selected from the group consisting of:
hydrogen;
unsubstituted lower alkyl;
lower alkyl substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3 and xe2x80x94NR13R14;
unsubstituted lower alkoxy;
lower alkoxy substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3, unsubstituted aryl or xe2x80x94NR13R14;
trihalomethyl;
unsubstituted alkenyl;
unsubstituted alkynyl;
unsubstituted aryl;
aryl substituted with one or more groups independently selected from the groups consisting of unsubstituted lower alkyl or lower alkyl substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3 and xe2x80x94NR13R14;
unsubstituted heteroalicyclic;
heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, xe2x80x94C(xe2x95x90O)H, xe2x80x94C(xe2x95x90O)xe2x80x94 (unsubstituted lower alkyl), hydroxy, unsubstituted alkoxy, alkoxy substituted with a group selected from the group consisting of halo, xe2x80x94C(xe2x95x90O)ORxe2x80x3 and xe2x80x94NR13R14;
unsubstituted aryloxy;
aryloxy substituted with a one or more groups independently selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, hydroxy and amino;
mercapto;
unsubstituted alkylthio;
unsubstituted arylthio;
arylthio substituted with one or more groups independently selected from the group consisting of halo, hydroxy or amino;
S-sulfonamido;
xe2x80x94C(xe2x95x90O)ORxe2x80x3;
Rxe2x80x3C(xe2x95x90O)Oxe2x80x94;
hydroxy;
cyano;
nitro;
halo;
C-amido;
N-amido;
amino; and,
xe2x80x94NR13R14, wherein
R13 is hydrogen and R14 is unsubstituted lower alkyl is another presently preferred embodiment of this invention.
A still further presently preferred embodiment of this invention is a compound in which:
R10, R11 and R12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, (CH2)rC(xe2x95x90O)ORxe2x80x3, xe2x80x94(CH2)rC(xe2x95x90O)Oxe2x88x92M+, halo, hydroxy, alkoxy, Rxe2x80x3C(xe2x95x90O)Oxe2x80x94, xe2x80x94C(xe2x95x90O))ORxe2x80x3, xe2x80x94C(xe2x95x90O)Oxe2x88x92M+, amino, C-amido, N-amido, nitro and xe2x80x94NR13R14.
A compound in which:
n is 1;
A, B, D and E are carbon;
R1 is hydrogen;
Z is oxygen;
R13 is hydrogen;
R14 is unsubstituted lower alkyl; and,
R10, R11 and R12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, xe2x80x94(CH2)C(xe2x95x90O)ORxe2x80x3, xe2x80x94(CH2)rC(xe2x95x90O)Oxe2x88x92M+, halo, hydroxy, alkoxy, Rxe2x80x3C(xe2x95x90O)Oxe2x80x94, xe2x80x94C(xe2x95x90O)ORxe2x80x3, xe2x80x94C(xe2x95x90O)Oxe2x88x92M+, amino, C-amido, N-amido, nitro and xe2x80x94NR13R14 is also a presently preferred embodiment of this invention.
A compound having the structural features in the paragraph immediately above wherein at least one of R10, R11 or R12 is selected from the group consisting of xe2x80x94C(xe2x95x90O)ORxe2x80x3, xe2x80x94C(xe2x95x90O)Oxe2x88x92M+, xe2x80x94(CH2)rC(xe2x95x90O)ORxe2x80x3 and xe2x80x94(CH2)rC(xe2x95x90O)Oxe2x88x92M+ is a presently preferred embodiment of this invention.
It is also a presently preferred embodiment of this invention that, in a compound having the structural features described in the paragraph immediately above this one, r of the xe2x80x94(CH2)rC(xe2x95x90O)ORxe2x80x3 or xe2x80x94(CH2)rC(xe2x95x90O)Oxe2x88x92 M+ group is 1 or 2.
Representative compounds of this invention are shown in Table 1. The compounds shown are presented by way of example only and are not to be construed as limiting the scope of this invention in any manner whatsoever.
1. Table 1 shows the chemical structures of exemplary compounds of this invention. The compound numbers correspond to the compound numbers in the Examples section, below. That is, the synthesis of compound 1 in Table 1 is Example 1 in the Examples section. These compounds are presented as examples only and are not to be construed as limiting the scope of this invention in any manner whatsoever.
2. Table 2 shows the results of biological assays of exemplary compounds of this invention. As above, the compound numbers is Table 2 correspond to the compound numbers in Table 1. The bioassays used are described in detail below. The results are given in terms of IC50, the micromolar (xcexcM) concentration of the compound being tested which effects a 50% change in the activity of a target PTK compared to the activity of the PTK in a control in which no compound of this invention is present. Specifically, the results shown indicate the concentration of the test compound needed to effect a 50% inhibition of the activity of the target PTK observed in the absence of a compound of this invention.
3. Table 3 shows the results of in vivo tests using the A375 sc cell line in the animal xenograft model described below. Compound 13 has the chemical structure shown in Table 1.
In yet another embodiment, this invention relates to a method for the modulation of the catalytic activity of PKs by contacting a PK with a compound of this invention or a physiologically acceptable salt or prodrug thereof.
A further embodiment of this invention is a method for identifying a compound which modulates the activity of a PK whichmethod consists of contacting a cell which the PK of interest with a compound and monitoring the effect of the compound on the cell.
By xe2x80x9cPKxe2x80x9d is meant RTKs, CTKs and STKs; i.e., the modulation of RTK, CTK and STK catalyzed signaling processes are contemplated by this invention.
The term xe2x80x9cmethodxe2x80x9d refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by, practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term xe2x80x9cmodulationxe2x80x9d or xe2x80x9cmodulatingxe2x80x9d refers to the alteration of the catalytic activity of RTKs, CTKs and STKs. In particular, modulating refers to the activation of the catalytic activity of RTKS, CTKs and STKS, preferably the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to which the RTK, CTK or STK is exposed or, more preferably, the inhibition of the catalytic activity of RTKs, CTKs and STKs.
The term xe2x80x9ccatalytic activityxe2x80x9d as used herein refers to the rate of phosphorylation of tyrosine under the influence, direct or indirect, of RTKs and/or CTKs or the phosphorylation of serine and threonine under the influence, direct or indirect, of STKs.
The term xe2x80x9ccontactingxe2x80x9d as used herein refers to bringing a compound of this invention and a target PK together in such a manner that the compound can affect the catalytic activity of the PK, either directly; i.e., by interacting with the kinase itself, or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent. Such xe2x80x9ccontactingxe2x80x9d can be accomplished in a test tube, a petri dish or the like. In a test tube, contacting may involve only a compound and a PK of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with the compound in that environment. In this context, the ability of a particular compound to affect a PK related disorder; i.e., the IC50, of the compound, defined below, can be determined before use of the compounds in vivo with more complex living organisms is attempted. For cells outside the organism, multiple methods exist, and are well-known to those skilled in the art, to get the PKs in contact with the compounds including, but not limited to, direct cell microinjection and numerous transmembrane carrier techniques.
By xe2x80x9cmonitoringxe2x80x9d or xe2x80x9cobservingxe2x80x9d is meant detecting the effect of contacting a compound with a cell expressing a particular PK.
The detected effect can be a change in cell phenotype, in the catalytic activity of a PK or a change in the interaction of a PK with a natural binding partner.
xe2x80x9cCell phenotypexe2x80x9d refers to the outward appearance of a cell or tissue or the biological function of the cell or tissue. Examples, without limitation, of a cell phenotype is cell size, cell growth, cell differentiation, cell proliferation, cell survival, apoptosis and nutrient uptake and use. Such phenotypic characteristics are detectable by techniques well-known in the art.
A xe2x80x9cnatural binding partnerxe2x80x9d refers to a polypeptide that binds to a particular PK in a cell. Natural binding partners can plan a role in propagating a signal in a PK-mediated signal transduction process. A change in the interaction of the natural binding partner with the PK can manifest itself as an increased or decreased concentration of the PK/natural binding partner complex resulting in a detectable change in the ability of the PK to mediate signal transduction.
RTK mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects on the extracellular microenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron 9:303-391.
It has been shown that tyrosine phosphorylation sites on growth factor receptors function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423; Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785); Songyang et al., 1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678. Several intracellular substrate proteins that associate with RTKs have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993, Cell, 72:767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993, Cell, 72:767-778. These observations suggest that the function of each RTK is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors as well as differentiation factor receptors.
STKs, being primarily cytosolic, affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process which initiates DNA synthesis and subsequent mitosis leading to cell proliferation.
Thus, PK signal transduction results in, among other responses, cell proliferation, differentiation, growth and metabolism. Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy and other disorders related to uncontrolled angiogenesis and/or vasculogenesis.
A precise understanding of the mechanism by which the compounds of this invention inhibit PKs is not required in order to practice the present invention. However, while not hereby being bound to any particular mechanism or theory, it is believed that the compounds interact with the amino acids in the catalytic region of PKs. PKs typically possess a bi-lobate structure wherein ATP appears to bind in the cleft between the two lobes in a region where the amino acids are conserved among PKs. Inhibitors of PKs are believed to bind by non-covalent interactions such as hydrogen bonding, van der Waals forces and ionic interactions in the same general region where the aforesaid ATP binds to the PKs. More specifically, it is thought that the 2-indolinone component of the compounds of this invention binds in the general space normally occupied by the adenine ring of ATP. Specificity of a particular molecule for a particular PK may then arise as the result of additional interactions between the various substituents on the 2-indolinone core and the amino acid domains specific to particular PKs. Thus, different indolinone substituents may contribute to preferential binding to particular PKs. The ability to select compounds active at different ATP (or other nucleotide) binding sites makes the compounds of this invention useful for targeting any protein with such a site; i.e., not only PKs but protein phosphatases as well. The compounds disclosed herein may thus have utility as in vitro assays for such proteins as well as exhibiting in vivo therapeutic effects through interaction with such proteins.
In another aspect, the protein kinase, the catalytic activity of which is modulated by contact with a compound of this invention, is a protein tyrosine kinase, more particularly, a receptor protein tyrosine kinase. Among the receptor protein tyrosine kinases whose catalytic activity can be modulated with a compound of this invention, or salt thereof, are, without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRxcex1, PDGFRxcex2, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
The protein tyrosine kinase whose catalytic activity is modulated by contact with a compound of this invention, or a salt or a prodrug thereof, can also be a non-receptor or cellular protein tyrosine kinase (CTK). Thus, the catalytic activity of CTKs such as, without limitation, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may be modulated by contact with a compound or salt of this invention.
Still another group of PKs which may have their catalytic activity modulated by contact with a compound of this invention are the serine-threonine protein kinases such as, without limitation, CDK2 and Raf.
In another aspect, this invention relates to a method for treating or preventing a PK related disorder by administering a therapeutically effective amount of a compound of this invention, or a salt or a prodrug thereof, to an organism.
In a further aspect, this invention relates to a method for treating or preventing a PK related disorder administering a therapeutically effective amount of a pharmacological composition of a compound of this invention, or a salt or prodrug thereof, to an organism.
As used herein, xe2x80x9cPK related disorder,xe2x80x9d xe2x80x9cPK driven disorder,xe2x80x9d and xe2x80x9cabnormal PK activityxe2x80x9d all refer to a condition characterized by inappropriate; i.e., under or, more commonly, over, PK catalytic activity, where the particular PK can be an RTK, a CTK or an STK. Inappropriate catalytic activity can arise as the result of either: (1) PK expression in cells which normally do not express PKs; (2) increased PK expression leading to unwanted cell proliferation, differentiation and/or growth; or, (3) decreased PK expression leading to unwanted reductions in cell proliferation, differentiation and/or growth. Over-activity of PKs refers to either amplification of the gene encoding a particular PK or production of a level of PK activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the PK increases, the severity of one or more of the symptoms of the cellular disorder increases). Underactivity is, of course, the converse, wherein the severity of one or more symptoms of a cellular disorder increase as the level of the PK decreases.
As used herein, the terms xe2x80x9cpreventxe2x80x9d, xe2x80x9cpreventingxe2x80x9d and xe2x80x9cpreventionxe2x80x9d refer to a method for barring an organism from acquiring a PK mediated cellular disorder in the first place.
As used herein, the terms xe2x80x9ctreatxe2x80x9d, xe2x80x9ctreatingxe2x80x9d and xe2x80x9ctreatmentxe2x80x9d refer to a method of alleviating or abrogating a PK mediated cellular disorder and/or its attendant symptoms. With regard particularly to cancer, these terms simply mean that the life expectancy of an individual affected with a cancer will be increased or that one or more of the symptoms of the disease will be reduced.
The term xe2x80x9corganismxe2x80x9d refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukariotic cell or as complex as a mammal, including a human being.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor; (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth; and/or, (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
This invention is therefore directed to compounds which modulate PK signal transduction by affecting the enzymatic activity of RTKs, CTKs and/or STKs, thereby interfering with the signals transduced by such proteins. More particularly, the present invention is directed to compounds which modulate RTK, CTK and/or STK mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinomas, sarcomas, including Kaposi""s sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Treatment or prevention of non-solid tumor cancers such as leukemia are also contemplated by this invention. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers and bone cancers.
Further examples, without limitation, of the types of disorders related to unregulated PK activity that the compounds described herein may be useful in preventing, treating and studying, are cell proliferative disorders, fibrotic disorders and metabolic disorders.
Cell proliferative disorders, which may be prevented, treated or further studied by the present invention include cancer, blood vessel proliferative disorders and mesangial cell proliferative disorders.
Blood vessel proliferative disorders refer to disorders related to abnormal vasculogenesis (blood vessel formation) and angiogenesis (spreading of blood vessels). While vasculogenesis and angiogenesis play important roles in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration, they also play a pivotal role in cancer development where they result in the formation of new capillaries needed to keep a tumor alive. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness.
Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, may also be treated or prevented by the methods of this invention.
Fibrotic disorders refer to the abnormal formation of extracellular matrices. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis. Another fibrotic disorder is atherosclerosis.
Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis as well such disorders as thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. PDGFR has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney International 43:47Sa54S.
As noted previously, PKs have been associated with cell proliferative disorders. Thus it is not surprising that some members of the RTK family have been associated with the development of cancer. Some of these receptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) are over-expressed in many tumors and/or persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor over-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci., 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070; Korc et al., supra; Akbasak and Suner-Akbasak et al., supra) have been demonstrated. For example, EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has been associated.with breast, ovarian, gastric, lung, pancreas and bladder cancer. PDGFR has been associated with glioblastoma and melanoma as well as lung, ovarian and prostate cancer. The RTK c-met has been associated with malignant tumor formation. For example, c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic, gastric and hepatocellular carcinomas and lymphomas. Additionally c-met has been linked to leukemia. Over-expression of the c-met gene has also been detected in patients with Hodgkins disease and Burkitts disease.
Flk/KDR has likewise been associated with a broad spectrum of tumors including, without limitation, mammary, ovarian and lung tumors as well as gliomas such as glioblastoma.
IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers. For example, IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). In addition, IGF-I, while integrally involved in the normal growth and differentiation of the nervous system, also appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478. The importance of IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stem cells of the bone marrow)) are stimulated to grow by IGF-I. Goldring and Goldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series of recent publications, Baserga suggests that IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga, 1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol., 14:4588-4595.
STKs have been implicated in many types of cancer including, notably, breast cancer (Cance, et al., Int. J. Cancer, 54:571-77 (1993)).
The association between abnormal PK activity and disease is not restricted to cancer. For example, RTKs have been associated with diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer""s disease, epidermal hyperproliferation, neuro-degenerative diseases, age-related macular degeneration and hemangiomas. For instance, EGFR is indicated in corneal and dermal wound healing. Defects in Insulin-R and IGF-1R are indicated in type-II diabetes mellitus. A more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., 1994, DNandP, 7:334-339.
As noted previously, not only RTKs but CTKs as well including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al., 1992, FASEB J., 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus could be expected, and have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed. For example, mutated src (v-src) has been demonstrated to be an oncoprotein (pp60v-src) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60c-src transmits oncogenic signals of many receptors. Over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60cXsrc, which is characteristic of malignant cells but absent in normal cells. On the other hand, mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.
Similarly, Zap70 has been connected with T-cell signaling which may have implications in autoimmune disorders.
STKs have been associated with inflamation, autoimmune disease, immunoresponses, and hyperproliferation disorders such as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.
PKs have also been implicated in embryo implantation. Thus, the compounds of this invention may provide an effective method of preventing such embryo implantation and thereby be useful as birth control agents.
Finally, both RTKs and CTKs are currently suspected as being involved in hyperimmune disorders. Thus, it is an aspect of this invention that protein kinase related cancers such as, without limitation, squamous cell carcinoma, astrocytoma, Kaposi""s sarcoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, small-cell lung cancer, glioma, colorectal cancer, genitourinary cancer and gasterointestinal cancer may be treated or prevented by administration to an organism of a therapeutically effective amount of a compound of this invention.
In a further aspect of this invention, non-cancer protein kinase related diorders such as, without limitation, diabetes, autoimmune disorder, immunological disorder, hyperproliferative disorder, restenosis, fibrosis, psoriasis, von Hippel-Lindau disease, osteoarthritis, rheumatoid arthritis, angiogenesis, inflammatory disorder and cardiovascular disease, may also be treated or prevented by the administration of a therapeutically effective amount of a compound of this invention to an organism.
Tables 2 and 3 show the activity of representative compounds of this invention against several of the above-described RTKs. Neither the compounds shown, the levels of activity indicated nor the specific RTKs affected are to be construed as limiting the scope of this invention in any manner whatsoever.
A compound of the present invention, a prodrug thereto or a physiologically acceptable salt of either the compound or its prodrug, can be administered as such to a human patient or in pharmacological compositions in which the foregoing materials are mixed with suitable carriers or excipient(s). Techniques for formulation and administration of drugs may be found in xe2x80x9cRemington""s Pharmaceutical Sciences,xe2x80x9d Mack Publishing Co., Easton, Pa., latest edition.
Routes of Administration.
As used herein, xe2x80x9cadministerxe2x80x9d or xe2x80x9cadministrationxe2x80x9d refers to the delivery of a compound, salt or prodrug of the present invention or of a pharamacological composition containing a compound, salt or prodrug of this invention to an organism for the purpose of prevention or treatment of a PK-related disorder.
Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.
Composition/Formulation.
Pharmacological compositions of the present invention may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmacological compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks"" solution, Ringer""s solution, or physiological saline buffer.
For transmucosal administration, penetrants appropriate to the barrier to be permeated ate used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores. Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmacological compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added in these formulations, also.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichloro tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating materials such as suspending, stabilizing and/or dispersing agents.
Pharmacological compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound. Additionally, suspensions of the active compounds may be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers and/or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. A compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharamcologically acceptable oil), with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt.
The pharmacological compositions herein also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the PK modulating compounds of the invention may be provided as physiologically acceptable salts wherein the claimed compound may form the negatively or the positively charged species. Examples of salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate, succinate wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid. Salts in which a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide (Ca(OH)2), etc.).
Dosage.
Pharmacological compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose; i.e., the modulation of PK activity or the treatment or prevention of a PK-related disorder.
More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PK activity). Such information can then be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 and the LD50 (both of which are discussed elsewhere herein) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient""s condition. (See e.g., Fingl, et al., 1975, in xe2x80x9cThe Pharmacological Basis of Therapeuticsxe2x80x9d, Ch. 1 p.1).
Dosage amount and interval may be adjusted individually to provide plasma levels of the active specie which are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage amount and interval.
The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Packaging.
The compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or of human or veterinary administration. Such notice, for example, may be of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
The compounds of this invention, as well as the precursor 2-oxindoles and cycloketones, may be synthesized using the procedure described below. Other approaches to the synthesis of the compounds and/or precursors to the compounds of this invention may become apparent to those skilled in the art based on the disclosures herein. Such alternate procedures are within the scope and spirit of this invention.
General Synthetic Procedure.
One to three equivalents of the appropriate 2-oxindole and one equivalent of the appropriate cycloketone are mixed together in water or an organic solvent such as, without limitation, C1-C10 alcohols (methanol, ethanol, octanol, etc.), ethylene glycol, cellosolve, diethyl ether, dichloromethane, chloroform, carbon tetrachloride, benzene, toluene, xylene, tetrahydrofuran, acetonitrile, dioxane, dimethylsulfoxide, dimethylformamide, dimethylacetamide, 1-methyl-2-pyrrolidine, pyridine and the like. Preferably the solvent is an organic solvent selected from the group consisting of dimethylsulfoxide, dimethylformamide, dimethylacetamide and N-methylpyrrolidine. Most preferably, the solvent is selected from dimethylformamide and dimethylacetamide. A molar excess of an inorganic or organic base is added. The base may be, without limitation, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium methoxide, sodium ethoxide, potassium t-butoxide, triethylamine, triethanolamine, piperidine, morpholine, pyrrolidine and the like. Preferably, the base is an organic base selected from the group consisting of triethylamine, piperidine, morpholine and pyrrolidine. Most preferably, the base is piperidine. The resulting solution or mixture is stirred at from about 20xc2x0 C. to about 200xc2x0 C. for from about 0.5 hour to about 100 hours at atmospheric pressure or in a sealed tube. The temperature is preferably about 90xc2x0 C. to about 175xc2x0 C., most preferably about 110xc2x0 C. to about 150xc2x0 C. The reaction time is preferably 1 hour to about 72 hours. The mixture is then brought to room temperature. Dilute aqueous inorganic acid (for example, without limitation, 1N hydrochloric acid or 1N sulfuric acid) is then added in an amount sufficient to neutralize the base. The resulting mixture is extracted with a water insoluble organic solvent such as without limitation, methylene chloride, diethyl ether, hexane, ethyl acetate, benzene or mixtures of solvents such as ethyl acetate/hexane. Preferably, the organic solvent is ethyl acetate. The organic solvent layer is separated, dried and evaporated to give the compound of this invention.